RESUMO
The wall proteome and the secretome of the fungal pathogen Candida albicans help it to thrive in multiple niches of the human body. Mass spectrometry has allowed researchers to study the dynamics of both subproteomes. Here, we discuss some major responses of the secretome to host-related environmental conditions. Three ß-1,3-glucan-modifying enzymes, Mp65, Sun41, and Tos1, are consistently found in large amounts in culture supernatants, suggesting that they are needed for construction and expansion of the cell wall ß-1,3-glucan layer and thus correlate with growth and might serve as diagnostic biomarkers. The genes ENG1, CHT3, and SCW11, which encode an endoglucanase, the major chitinase, and a ß-1,3-glucan-modifying enzyme, respectively, are periodically expressed and peak in M/G1. The corresponding protein abundances in the medium correlate with the degree of cell separation during single-yeast-cell, pseudohyphal, and hyphal growth. We also discuss the observation that cells treated with fluconazole, or other agents causing cell surface stress, form pseudohyphal aggregates. Fluconazole-treated cells secrete abundant amounts of the transglucosylase Phr1, which is involved in the accumulation of ß-1,3-glucan in biofilms, raising the question whether this is a general response to cell surface stress. Other abundant secretome proteins also contribute to biofilm formation, emphasizing the important role of secretome proteins in this mode of growth. Finally, we discuss the relevance of these observations to therapeutic intervention. Together, these data illustrate that C. albicans actively adapts its secretome to environmental conditions, thus promoting its survival in widely divergent niches of the human body.
Assuntos
Adaptação Fisiológica , Candida albicans/fisiologia , Meio Ambiente , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Animais , Glicosilfosfatidilinositóis/metabolismo , HumanosRESUMO
Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.
Assuntos
Candida/fisiologia , Candida/patogenicidade , Evolução Molecular , Genoma Fúngico/genética , Reprodução/genética , Candida/classificação , Candida/genética , Códon/genética , Sequência Conservada , Diploide , Genes Fúngicos/genética , Meiose/genética , Polimorfismo Genético , Saccharomyces/classificação , Saccharomyces/genética , Virulência/genéticaRESUMO
Bionumbers and bioestimates are valuable tools in biological research. Here we focus on cell wall-related bionumbers and bioestimates of the budding yeast Saccharomyces cerevisiae and the polymorphic, pathogenic fungus Candida albicans. We discuss the linear relationship between cell size and cell ploidy, the correlation between cell size and specific growth rate, the effect of turgor pressure on cell size, and the reason why using fixed cells for measuring cellular dimensions can result in serious underestimation of in vivo values. We further consider the evidence that individual buds and hyphae grow linearly and that exponential growth of the population results from regular formation of new daughter cells and regular hyphal branching. Our calculations show that hyphal growth allows C. albicans to cover much larger distances per unit of time than the yeast mode of growth and that this is accompanied by strongly increased surface expansion rates. We therefore predict that the transcript levels of genes involved in wall formation increase during hyphal growth. Interestingly, wall proteins and polysaccharides seem barely, if at all, subject to turnover and replacement. A general lesson is how strongly most bionumbers and bioestimates depend on environmental conditions and genetic background, thus reemphasizing the importance of well-defined and carefully chosen culture conditions and experimental approaches. Finally, we propose that the numbers and estimates described here offer a solid starting point for similar studies of other cell compartments and other yeast species.
Assuntos
Candida albicans/citologia , Parede Celular/metabolismo , Saccharomyces cerevisiae/citologia , Candida albicans/metabolismo , Candida albicans/fisiologia , Processos de Crescimento Celular , Parede Celular/química , Proteínas Fúngicas/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologiaRESUMO
In Saccharomyces cerevisiae, the vacuolar protein sorting complexes Vps51/52/53/54 and Vps15/30/34/38 are essential for efficient endosome-to-Golgi complex retrograde transport. Here we investigated the function of Vps15 and Vps51, representative members of these complexes, in the stress resistance, host cell interactions, and virulence of Candida albicans. We found that C. albicans vps15Δ/Δ and vps51Δ/Δ mutants had abnormal vacuolar morphology, impaired retrograde protein trafficking, and dramatically increased susceptibility to a variety of stressors. These mutants also had reduced capacity to invade and damage oral epithelial cells in vitro and attenuated virulence in the mouse model of oropharyngeal candidiasis. Proteomic analysis of the cell wall of the vps51Δ/Δ mutant revealed increased levels of the Crh11 and Utr2 transglycosylases, which are targets of the calcineurin signaling pathway. The transcript levels of the calcineurin pathway members CHR11, UTR2, CRZ1, CNA1, and CNA2 were elevated in the vps15Δ/Δ and vps51Δ/Δ mutants. Furthermore, these strains were highly sensitive to the calcineurin-specific inhibitor FK506. Also, deletion of CHR11 and UTR2 further increased the stress susceptibility of these mutants. In contrast, overexpression of CRH11 and UTR2 partially rescued their defects in stress resistance, but not host cell interactions. Therefore, intact retrograde trafficking in C. albicans is essential for stress resistance, host cell interactions, and virulence. Aberrant retrograde trafficking stimulates the calcineurin signaling pathway, leading to the increased expression of Chr11 and Utr2, which enables C. albicans to withstand environmental stress.
Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Estresse Fisiológico , Proteína VPS15 de Distribuição Vacuolar/metabolismo , Animais , Calcineurina/genética , Calcineurina/metabolismo , Inibidores de Calcineurina , Candida albicans/genética , Candida albicans/patogenicidade , Candidíase Bucal/microbiologia , Proteínas Fúngicas/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Transporte Proteico , Tacrolimo/farmacologia , Proteína VPS15 de Distribuição Vacuolar/genética , Virulência/genéticaRESUMO
The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using (15)N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the ß-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.
Assuntos
Candida albicans/fisiologia , Parede Celular/metabolismo , Candida albicans/citologia , Quitina/metabolismo , Quitinases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Estresse FisiológicoRESUMO
The opportunistic fungal pathogen Candida albicans has developed various ways to overcome iron restriction in a mammalian host. Using different surface proteins, among them membrane- and wall-localized glycosylphosphatidylinositol (GPI) proteins, it can exploit iron from host haemoglobin, ferritin and transferrin. Culturing C. albicans in rich medium supplemented with the ferrous iron chelator bathophenanthroline disulfonic acid or in the minimal medium yeast nitrogen base resulted in a strong decrease of the iron content of the cells. MS analysis of the changes in the wall proteome of C. albicans upon iron restriction showed a strong increase in the levels of the GPI-modified adhesin Als3, which also serves as a ferritin receptor, and of the GPI-modified CFEM (common in fungal extracellular membranes) domain-containing proteins Csa1, Pga7, Pga10, and Rbt5. The wall levels of the GPI-modified proteins Hyr1, the adhesin Als4 and the copper- and zinc-containing superoxide dismutase Sod4 also strongly increased, whereas the levels of Tos1 (a non-GPI protein) and the GPI-modified adhesin Als2 strongly decreased. Strikingly, peptides derived from the CFEM domain of the haem-binding proteins Csa1, Pga10 and Rbt5 were capable of forming iron adduct ions during MS analysis, consistent with a key role of this domain in haem binding.
Assuntos
Candida albicans/química , Candida albicans/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Proteínas Fúngicas/análise , Ferro/metabolismo , Proteoma/análise , Candida albicans/crescimento & desenvolvimento , Meios de Cultura/química , Perfilação da Expressão Gênica , Espectrometria de Massas , Proteínas de Membrana/análiseRESUMO
The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose-grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate-grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose-grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate-grown cells. We identified mating and pheromone-regulated proteins that were exclusive to lactate-grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa-specific and other niche-specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.
Assuntos
Candida albicans/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Proteoma/metabolismo , Antifúngicos/farmacologia , Biofilmes , Candida albicans/metabolismo , Candida albicans/fisiologia , Parede Celular/química , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Ácido Láctico/metabolismo , Ácido Láctico/farmacologia , Testes de Sensibilidade Microbiana , Pressão Osmótica , Fenótipo , Proteoma/efeitos dos fármacos , Estresse FisiológicoRESUMO
Fluconazole is a commonly used antifungal drug that inhibits Erg11, a protein responsible for 14α-demethylation during ergosterol synthesis. Consequently, ergosterol is depleted from cellular membranes and replaced by toxic 14α-methylated sterols, which causes increased membrane fluidity and drug permeability. Surface-grown and planktonic cultures of Candida albicans responded similarly to fluconazole at 0.5 mg/liter, showing reduced biomass formation, severely reduced ergosterol levels, and almost complete inhibition of hyphal growth. There was no evidence of cell leakage. Mass spectrometric analysis of the secretome showed that its composition was strongly affected and included 17 fluconazole-specific secretory proteins. Relative quantification of (14)N-labeled query walls relative to a reference standard mixture of (15)N-labeled yeast and hyphal walls in combination with immunological analysis revealed considerable fluconazole-induced changes in the wall proteome as well. They were, however, similar for both surface-grown and planktonic cultures. Two major trends emerged: (i) decreased incorporation of hypha-associated wall proteins (Als3, Hwp1, and Plb5), consistent with inhibition of hyphal growth, and (ii) increased incorporation of putative wall repair-related proteins (Crh11, Pga4, Phr1, Phr2, Pir1, and Sap9). As exposure to the wall-perturbing drug Congo red led to a similar response, these observations suggested that fluconazole affects the wall. In keeping with this, the resistance of fluconazole-treated cells to wall-perturbing compounds decreased. We propose that fluconazole affects the integrity of both the cellular membranes and the fungal wall and discuss its potential consequences for antifungal therapy. We also present candidate proteins from the secretome for clinical marker development.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Parede Celular/metabolismo , Fluconazol/farmacologia , Proteínas Fúngicas/metabolismo , Hifas/efeitos dos fármacos , Sequência de Aminoácidos , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Parede Celular/efeitos dos fármacos , Análise de Fourier , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/químicaRESUMO
Recently, the food yeast Candida utilis has emerged as an excellent host for production of heterologous proteins. Since secretion of the recombinant product is advantageous for its purification, we characterized the secreted proteome of C. utilis. Cells were cultivated to the exponential or stationary growth phase, and the proteins in the medium were identified by MS. In parallel, a draft genome sequence of C. utilis strain DSM 2361 was determined by massively parallel sequencing. Comparisons of protein and coding sequences established that C. utilis is not a member of the CUG clade of Candida species. In total, we identified 37 proteins in the culture solution, 17 of which were exclusively present in the stationary phase, whereas three proteins were specific to the exponential growth phase. Identified proteins represented mostly carbohydrate-active enzymes associated with cell wall organization, while no proteolytic enzymes and only a few cytoplasmic proteins were detected. Remarkably, cultivation in xylose-based medium generated a protein pattern that diverged significantly from glucose-grown cells, containing the invertase Inv1 as the major extracellular protein, particularly in its highly glycosylated S-form (slow-migrating). Furthermore, cultivation without ammonium sulfate induced the secretion of the asparaginase Asp3. Comparisons of the secretome of C. utilis with those of Kluyveromyces lactis and Pichia pastoris, as well as with those of the human fungal pathogens Candida albicans and Candida glabrata, revealed a conserved set of 10 and six secretory proteins, respectively.
Assuntos
Candida/metabolismo , Proteoma/metabolismo , Candida/genética , Candida/crescimento & desenvolvimento , Carbono/metabolismo , Códon , Microbiologia de Alimentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Humanos , Nitrogênio/metabolismo , Filogenia , Proteoma/genética , RNA Ribossômico 18S/genética , Análise de Sequência de DNARESUMO
The ability of Candida albicans to switch from yeast to hyphal growth is essential for its virulence. The walls and especially the covalently attached wall proteins are involved in the primary host-pathogen interactions. Three hyphal induction methods were compared, based on fetal calf serum, the amino sugar N-acetylglucosamine (GlcNAc) and the mammalian cell culture medium Iscove's modified Dulbecco's medium (IMDM). GlcNAc and IMDM were preferred, allowing stable hyphal growth over a prolonged period without significant reversion to yeast growth and with high biomass yields. We employed Fourier transform-MS combined with a (15)N-metabolically labelled reference culture as internal standard for relative quantification of changes in the wall proteome upon hyphal induction. A total of 21 wall proteins were quantified. Our induction methods triggered a similar response characterized by (i) a category of wall proteins showing strongly increased incorporation levels (Als3, Hwp2, Hyr1, Plb5 and Sod5), (ii) another category with strongly decreased levels (Rhd3, Sod4 and Ywp1) and (iii) a third one enriched for carbohydrate-active enzymes (including Cht2, Crh11, Mp65, Pga4, Phr1, Phr2 and Utr2) and showing only a limited response. This is, to our knowledge, the first systematic, quantitative analysis of the changes in the wall proteome of C. albicans upon hyphal induction. Finally, we propose new wall-protein-derived candidates for vaccine development.
Assuntos
Candida albicans/química , Candida albicans/crescimento & desenvolvimento , Parede Celular/química , Proteínas Fúngicas/análise , Regulação Fúngica da Expressão Gênica , Hifas/química , Hifas/crescimento & desenvolvimento , Meios de Cultura/química , Humanos , Proteoma/análise , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The mucosal layers colonized by the pathogenic fungus Candida albicans differ widely in ambient pH. Because the properties and functions of wall proteins are probably pH dependent, we hypothesized that C. albicans adapts its wall proteome to the external pH. We developed an in vitro system that mimics colonization of mucosal surfaces by growing biomats at pH 7 and 4 on semi-solid agarose containing mucin as the sole nitrogen source. The biomats expanded radially for at least 8 days at a rate of ~30 µm h(-1). At pH 7, hyphal growth predominated and growth was invasive, whereas at pH 4 only yeast and pseudohyphal cells were present and growth was noninvasive. Both qualitative mass spectrometric analysis of the wall proteome by tandem mass spectrometry and relative quantification of individual wall proteins (pH 7/pH 4), using Fourier transform mass spectrometry (FT-MS) and a reference mixture of (15)N-labelled yeast and hyphal walls, identified similar sets of >20 covalently linked wall proteins. The adhesion proteins Als1 and Als3, Hyr1, the transglucosidase Phr1, the detoxification enzyme Sod5 and the mammalian transglutaminase substrate Hwp1 (immunological detection) were only present at pH 7, whereas at pH 4 the level of the transglucosidase Phr2 was >35-fold higher than at pH 7. Sixteen out of the 22 proteins identified by FT-MS showed a greater than twofold change. These results demonstrate that ambient pH strongly affects the wall proteome of C. albicans, show that our quantitative approach can give detailed insights into the dynamics of the wall proteome, and point to potential vaccine targets.
Assuntos
Candida albicans/química , Candida albicans/fisiologia , Parede Celular/química , Proteínas Fúngicas/análise , Regulação Fúngica da Expressão Gênica , Proteoma/análise , Estresse Fisiológico , Concentração de Íons de Hidrogênio , Espectroscopia de Infravermelho com Transformada de Fourier , Espectrometria de Massas em TandemRESUMO
The covalently linked wall proteins of Saccharomyces cerevisiae and Candida albicans and to a lesser extent of Candida glabrata have been extensively studied. Here we describe some of their main structural features and discuss their conservation in other ascomycetous fungi. We also discuss the hybrid nature of many wall proteins and the frequent occurrence of families of wall proteins with a common multi-domain structure. Finally, some quantitative data regarding wall proteins are presented.
Assuntos
Ascomicetos/química , Ascomicetos/ultraestrutura , Parede Celular/química , Parede Celular/ultraestrutura , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Ascomicetos/genética , Parede Celular/genética , Proteínas Fúngicas/metabolismo , Modelos Biológicos , Modelos Químicos , ProteômicaRESUMO
The pathogenic fungus Candida albicans secretes a considerable number of hydrolases and other proteins. In-depth studies of the C. albicans secretome could thus provide new candidates for diagnostic markers and vaccine development. We compared various growth conditions differing in pH, temperature and the presence of the hyphal inducer N-acetylglucosamine. The polypeptide content of the growth media was ca. 0.1-0.2% of the total biomass. Using LC-tandem mass spectrometry, we identified 44 secretory proteins, the transmembrane protein Msb2, six secretory pathway-associated proteins and 28 predicted cytosolic proteins. Many secretory proteins are wall-related, suggesting that their presence in the growth medium is at least partially due to accidental release from the walls. Als3, Csa2, Rbt4, Sap4 and Sap6 were enriched in the medium of hyphal cultures; Bgl2, Cht3, Dag7, Eng1, Pir1, Rbe1, Scw11, Sim1/Sun42, Xog1 and Ywp1 in the medium of yeast cells; and Plb4.5 in pH 4 medium. Seven proteins (Cht3, Mp65, Orf19.5063/Coi1, Scw11, Sim1, Sun41 and Tos1) were consistently present under all conditions tested. These observations indicate that C. albicans tightly regulates its secretome. Mp65, Sun41, and Tos1 were each predicted to contain at least one highly immunogenic peptide. In total, we identified 29 highly immunogenic peptides originating from 18 proteins, including two members of the family of secreted aspartyl proteases. Fifty-six peptides were identified as proteotypic and will be useful for quantification purposes. In summary, the number of identified secretory proteins in the growth medium has been substantially extended, and growth conditions strongly affect the composition of the secretome.
Assuntos
Candida albicans/metabolismo , Meios de Cultura/química , Proteínas Fúngicas/análise , Proteínas Fúngicas/metabolismo , Proteoma/análise , Antígenos de Fungos/análise , Antígenos de Fungos/imunologia , Antígenos de Fungos/metabolismo , Candida albicans/crescimento & desenvolvimento , Cromatografia Líquida , Proteínas Fúngicas/imunologia , Humanos , Espectrometria de Massas em TandemRESUMO
In many ascomycetous yeasts, the cell wall is composed of two main types of macromolecules: (a) polysaccharides, with a high content of beta-1,6- and beta-1,3-linked glucan chains and minor amounts of chitin; and (b) cell wall proteins of different types. Synthesis and maintenance of these macromolecules respond to environmental changes, which are sensed by the cell wall integrity (CWI) signal transduction pathway. We here present a first systematic analysis of the cell wall composition of the milk yeast, Kluyveromyces lactis. Electron microscopic analyses revealed that exponentially growing cells of K. lactis supplied with glucose as a carbon source have a wall thickness of 64 nm, as compared to 105 nm when growing on 3% ethanol. Despite their increased wall thickness, ethanol-grown cells were more sensitive to the presence of zymolyase in the growth medium. Mass spectrometric analysis identified 22 covalently linked cell wall proteins, including 19 GPI-modified proteins and two Pir wall proteins. Importantly, the composition of the cell wall glycoproteome depended on carbon source and growth phase. Our results clearly illustrate the dynamic nature of the cell wall of K. lactis and provide a firm base for studying its regulation.
Assuntos
Parede Celular/química , Proteínas Fúngicas/análise , Glicoproteínas/análise , Kluyveromyces/química , Proteoma/análise , Carbono/metabolismo , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Meios de Cultura/química , Etanol/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Glucose/metabolismo , Kluyveromyces/metabolismo , Kluyveromyces/ultraestrutura , Espectrometria de Massas , Microscopia EletrônicaRESUMO
The glycosylphosphatidylinositol-modified protein Rhd3/Pga29 of the human pathogen Candida albicans belongs to a family of cell wall proteins that are widespread among Candida species but are not found in other fungi. Pga29 is covalently linked to the beta-1,3-glucan framework of the cell wall via beta-1,6-glucan. It is a small and abundant O-glycosylated protein and requires the protein-O-mannosyl transferase Pmt1 for glycosylation. Furthermore, Pga29 is strongly expressed in yeast cells but is downregulated in hyphae. Removal of the PGA29 gene in C. albicans leads to a significant reduction of cell wall mannan; however, Pga29 does not seem to have a major role in maintaining cell wall integrity. In addition, adhesion capacity and hyphae formation appear normal in pga29 deletion mutants. Importantly, the pga29 deletion mutant is less virulent, and infection of reconstituted human epithelium with the pga29 mutant results in a diminished induction of proinflammatory cytokines, such as GM-CSF, TNF, IL-6 and IL-8. We propose that the reduced virulence of the pga29 mutant is a consequence of altered surface properties, resulting in altered fungal recognition.
Assuntos
Candida albicans/química , Candida albicans/patogenicidade , Parede Celular/química , Proteínas Fúngicas/análise , Proteínas Fúngicas/fisiologia , Fatores de Virulência/análise , Fatores de Virulência/fisiologia , Citocinas/metabolismo , Células Epiteliais/microbiologia , Proteínas Fúngicas/genética , Deleção de Genes , Glicoproteínas/análise , Glicoproteínas/genética , Glicoproteínas/fisiologia , Humanos , Virulência , Fatores de Virulência/genéticaRESUMO
The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.
Assuntos
Aspergillus niger/genética , Mapeamento Cromossômico , Cromossomos Fúngicos/genética , Genoma Fúngico/genética , Proteínas de Plantas/genética , Análise de Sequência de DNA/métodos , Sequência de Bases , Dados de Sequência MolecularRESUMO
The manifold functions of fungal wall glycoproteins include maintenance of cell wall integrity, homotypic and heterotypic adhesion, biofilm formation, acquisition of iron and sterols, protein degradation and coping with oxidative stress. Transcriptome studies indicate that the expression levels of most cell wall glycoproteins can vary widely and are tightly controlled. However, owing to their complex and variable glycosylation, fungal wall glycoproteins are difficult to analyze using traditional proteomics approaches. Recent advances in mass spectrometry-based proteomics have enabled rapid and sensitive identification and quantitation of fungal wall glycoproteins; this will be particularly useful for studying the dynamics of the subproteome of fungal wall glycoproteins, and for the development of novel vaccines and diagnostic tools.
Assuntos
Parede Celular/química , Proteínas Fúngicas/análise , Fungos/química , Glicoproteínas/análise , Espectrometria de Massas , Proteômica/métodosRESUMO
Yeast cell wall-associated, lectin-like adhesins form large families that mediate flocculation and host cell recognition. The glycan specificity of individual adhesins is largely unknown. Zupancic et al. (this issue of Molecular Microbiology) used glycan microarrays to compare the glycan-binding characteristics of individual adhesins (Epa proteins) of the pathogenic yeast Candida glabrata produced in the non-adherent yeast Saccharomyces cerevisiae. By sequence swapping between the conserved PA14 domains of two related Epa proteins, they identified a pentapeptide that determines binding specificity and cell adherence and is located on a surface loop of the known crystal structure of the anthrax toxin PA14 domain.
Assuntos
Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Glucanos/metabolismo , Sítios de Ligação , Candida glabrata/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Proteínas Fúngicas/química , Estrutura Terciária de ProteínaRESUMO
Knowledge of the mechanisms underlying cell wall biosynthesis in Aspergillus spp. is of high relevance to medicine and food safety, and for biotechnological applications. The cell wall of Aspergillus nidulans is composed of galactomannoproteins, 1,3-alpha-glucan, beta-glucans, and chitin. Here, we present a comprehensive inventory of the cell wall-related genes in A. nidulans. This includes glycan-synthetic and glycan-processing enzymes, spore wall maturation enzymes, GPI-anchor processing enzymes, GPI proteins and hydrophobins, and signaling proteins of the cell wall integrity pathway. All major known fungal cell wall-related genes are represented, except for Pir-CWPs. Importantly, we have identified a gene product that is possibly involved in the synthesis of 1,3/1,4-beta-glucan, and we propose that four predicted GPI proteins, a mixed-linked beta-glucanase and three amylase-like alpha-glucanases, may have transglucosidic activities pertaining to the processing of 1,3/1,4-beta-glucan and 1,3/1,4-alpha-glucan, respectively. We further present an updated survey of putative GPI proteins. Finally, we present mass spectrometric evidence suggesting the presence of at least twelve covalently-linked cell wall proteins in the hyphal wall of A. nidulans, including ten predicted GPI proteins, most of which belong to carbohydrate-active enzyme families that are also found in the walls of ascomycetous yeasts.
Assuntos
Aspergillus nidulans/genética , Parede Celular/genética , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Glicosilfosfatidilinositóis/genética , Aspergillus nidulans/metabolismo , Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espectrometria de Massas , Polissacarídeos/metabolismo , Esporos FúngicosRESUMO
The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.